Abstract 497: Regulation of CSC lineage and stemness maintenance in progressive neuroblastoma: RD3, a MVP in the league
Authors: Natarajan Aravindan, Dinesh Babu Somasundaram
Journal: Cancer Research (2022)

Abstract Our studies recognized that the retinal degeneration protein 3 (RD3) is constitutively expressed in human adult and fetal tissues beyond retina. Further, we showed that transcriptional/translational loss of RD3 highly associated with advanced disease stage and poor clinical outcomes in neuroblastoma (NB) patients. We identified that RD3 loss is instrumental in NB pathogenesis and, more importantly defined its acquired loss in progressive NB after intensive multimodal clinical therapy. Herein, we investigated the mechanisms, how acquired loss of RD3 orchestrates NB progression. Cell lines derived during diagnosis from stage 4 NB patients off primary (CHLA-15) or metastatic (CHLA-42, SH-SY5Y) site, those with constitutive RD3 expression was used as parental controls. Analogous clones of stable RD3 knockouts were developed (shRNA-RNAi), validated (immunoblotting) and were assayed for whole genome transcriptomic modifications using high-throughput Illumina transcriptome RNA-Seq. Differential gene expression analysis with stringent criteria (log2 fold change) coupled with false discovery rate calculation clearly identified 575 conserved (across three independent cell-lines) genetic determinants that could translate RD3-loss associated disease progression. IPA-functional bioinformatics identified regulation of cellular-lineage directed (stem-cell pluripotency, EMT, Senescence, epithelial adherens junction etc.) signaling coupled with select tumor evolution (NFκB, GPCR, mTOR, Wnt/β-catenin, stat3 etc.) signaling. Individual gene QPCR validation of experimentally observed 13 lead (mean FDR &amp;gt;7.0) candidates affirmed RNA-Seq outcomes. Actuated FAM129A, HAS2, ICAM1, S100A11, TSPAN8, HLA-F, ASIC4, AXL, VCAM1, KLHDC7B, MAGEL2 and, repressed tumor suppressor FOXN4 and NB differentiation factor ROBO2 in RD3-KOs recognized diverted cell lineage (EMT, poor differentiation, stimulated CSCs, CSC stemness maintenance), drug resistance, immune modulation/evasion, and tumor progression (clonal selection and expansion, growth and malignancy, invasion and migration, angiogenesis). For the first time, these results demonstrate the RD3-loss dependent transcriptomic addiction of disease evolution drivers in NB cells. Further, the outcomes imply that RD3 directly regulates key CSC cell lineage and tumor progression/dissemination signaling events. Current investigations are appropriately directed to unveil the functional mechanism(s) that could benefit salvage therapy for progressive NB. Funding: This work was partially or in full, funded by Oklahoma Center for the Advancement of Science and Technology, OCAST-HR19-04; National Institutes of Health, NIH-P20GM103639 and NIH-NCI- Cancer Center Support Grant P30CA225520 Citation Format: Natarajan Aravindan, Dinesh Babu Somasundaram. Regulation of CSC lineage and stemness maintenance in progressive neuroblastoma: RD3, a MVP in the league [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 497.